Major efforts are underway to sequence whole genomes of a wide array of organisms and to determine the identities of all expressed proteins, yet the fundamental principles relating primary sequences of proteins to their three-dimensional structures remain incompletely understood. We propose experiments to elucidate the mechanism by which a representative of the intracellular lipid-binding protein (iLBP) family, cellular retinoic acid-binding protein I (CRABP I), adopts its native fold, with a goal of determining how these beta-barrel proteins successfully fold and avoid competing aggregation processes. We seek, as well, to extract general principles about beta-sheet folding and folding beta-barrels from the behavior of this important family. The iLBP family is widespread in eukaryotic cells and mediates critical functions, such as energy metabolism, signaling, and transcriptional regulation of differentiation. In addition to developing a full picture of the energy landscape for the folding of CRABP I in vitro, we propose new experiments to explore how CRABP I folds during its biosynthesis. This new direction of research seeks to fill the remarkable void in current understanding of the mechanism of folding in the cell. In the second major focus of this grant for the next project period, we will extend our studies on the mechanism of action of Hsp70 molecular chaperones. This ubiquitous family of chaperones carries out several related functions in the cell, all based on their ability to bind hydrophobic regions of polypeptide chains in a nucleotide-dependent manner. Their functions include facilitation of protein folding, disassembly of molecular complexes, protein translocation across membranes, protein degradation, and responses to stresses such as heat shock. We will test our emerging hypothesis that ligand-induced interdomain allostery in Hsp70 proteins relies on changes in the dynamics and intradomain stability in these two domain proteins. We will map in detail the conformational signal transduction pathway of the E. coli Hsp70, DnaK. We will also compare the modes of substrate binding by the two Hsp70s: DnaK from E. coli, and BiP from the eukaryotic endoplasmic reticulum. An increasing number of diseases has been associated with mistakes in protein folding or inadequacies of chaperone function (e.g., p53-based cancers, Alzheimer's, Huntington's, Parkinson's, cystic fibrosis, BSE), and enhanced understanding of both will aid design of therapeutic strategies.